Project Summary/Abstract During normal digestion, the vagus nerve sends both relaxation signals to the stomach to accommodate ingested food and contraction signals to triturate as well as passage food into the small intestines. These opposing functions are mediated by the same brain region, the dorsal motor nucleus of the vagus (DMV), but the mechanism by which vagal efferents deriving from the same anatomical brain structure exert different effects is poorly understood. While previous studies have led to the well-accepted postulate that the DMV contains separate functional units (i.e. labeled lines), these functional units have yet to be defined by gene expression and consequently there are no known recombinase driver mice that allow for selective access to the ?labeled lines? of vagal motor communication. Thus, it is unknown how many DMV labeled lines exist and what their respective roles are in digestion. By identifying genetically defined DMV subpopulations that control gastric functions, we can test models for how the DMV subtypes exert different effects. One possibility is that the DMV labeled lines exert different effects by engaging distinct postganglionic targets. In this model, the DMV neurons that drive stomach contraction engage cholinergic enteric neurons and those that relax the stomach activate enteric neurons that release nitric oxide. Evidence for this model, however, is inconclusive due to the lack recombinase driver mice for genetic access to the DMV subtypes. To identify DMV subtypes by their gene expression profiles, our lab has used single-nucleus RNA sequencing to transcriptionally profile, at the single cell level, cholinergic (Chat+) parasympathetic preganglionic motor neurons in the DMV. The study uncovered 9 subtypes of Chat+ DMV neurons and identified marker genes for genetic access. In Aim 1, we will identify and obtain genetic access to the DMV subtypes that project to the stomach. We will assemble or generate recombinase driver mice for each DMV subtype and use a recombinase-dependent adeno- associated virus expressing an anterograde tracer to map axon projections. In Aim 2, we will characterize the function of stomach-projecting DMV subtypes using optogenetics to manipulate the activity of each DMV subtype and assays to measure physiological responses (e.g. gastric pressure, acid secretion, gastrin secretion). In Aim 3, we will determine the downstream targets of the DMV subtypes that project to the stomach using channelrhodopsin-2-assisted circuit mapping (CRACM). We will visualize cholinergic enteric neurons using the existing Chat-GFP transgenic mouse line and nitrergic enteric neurons using Nos1-GFP mice that we will generate. In summary, this proposal will parse out the DMV ?labeled lines? that control gastric function and provide recombinase driver mice for genetic access to each DMV subtype. Importantly, having recombinase driver mice that provide selective access to each DMV ?labeled line? will allow for unprecedented studies that will significantly advance the field of vagal motor control over digestion and could provide groundbreaking insights on a broad range of clinical conditions.